A novel pH sensitive theranostic PLGA nanoparticle for boron neutron capture therapy in mesothelioma treatment

This study aims to develop poly lactic-co-glycolic acid (PLGA) nanoparticles with an innovative imaging-guided approach based on Boron Neutron Capture Therapy for the treatment of mesothelioma. The herein-reported results demonstrate that PLGA nanoparticles incorporating oligo-histidine chains and the dual Gd/B theranostic agent AT101 can successfully be exploited to deliver a therapeutic dose of boron to mesothelioma cells, significantly higher than in healthy mesothelial cells as assessed by ICP-MS and MRI. The selective release is pH responsive taking advantage of the slightly acidic pH of the tumour extracellular environment and triggered by the protonation of imidazole groups of histidine. After irradiation with thermal neutrons, tumoral and healthy cells survival and clonogenic ability were evaluated. Obtained results appear very promising, providing patients affected by this rare disease with an improved therapeutic option, exploiting PLGA nanoparticles.

www.nature.com/scientificreports/ this therapy can combine low-energy thermal neutron irradiation with the presence of boron-containing agents in the targeted pathological tissues. Neutrons are captured by non-radioactive 10 B, triggering a decaying nuclear reaction releasing alpha particles and 7 Li, causing a high biological damage in approximately 10 µm of diameter, which is less than the average diameter of a mammalian cell. Therefore, administering boron and producing alpha radiation selectively in diseased cells, NCT can kill the pathological ones while sparing the surrounding healthy tissues. These characteristics make BNCT a promising treatment for diffused metastasis and infiltrating tumours such as mesothelioma, that cannot be treated or are resistant to methods usually applicable in a localized tumour mass, such as conventional radiotherapy or surgery 14 . BNCT has been applied to skin melanoma, brain, head and neck tumours, and a large amount of clinical data has been collected through the different clinical trials (I/ II phase) carried out in Japan, USA, Netherlands, Sweden, Finland, Argentina, and Taiwan 15,16 . Nakamura and co-workers recently developed a hyaluronic acid containing sodium mercaptoundecahydro-closo-dodecaborate (BSH) specifically delivered to MM in preclinical mouse models 17 . Moreover, in 2006, a small number of MM patients were safely treated with BNCT in Japan, achieving significant palliation of the symptoms 18 . However, to achieve BNCT efficacy, the boron carriers have to respect different conditions: (i) low systemic toxicity; (ii) high selectivity for tumour cells; (iii) long half-life within the tumour for the duration of the treatment. It has been estimated that approximately 10-30 μg of B per gram of tumour mass are needed to attain an effective treatment, using a tolerable irradiation time and a suitable neutron source 19 . The two compounds currently used in clinical trials are p-borono-l-phenylalanine (BPA) (used in trials for glioblastoma, head and neck cancer, and melanoma) and BSH (designed for brain tumour treatments). These agents yield a tumour to normal tissue boron concentration ratio between 3 and 6, allowing a safe and quite effective treatment. However, it is a widespread opinion in the scientific community that a wider clinical application of BNCT will be possible when the uptake in targeting tumour cells will be further improved 19 . As an example of a successful targeting strategy, Low Density Lipoproteins (LDL) loaded with AT101, have been proposed by our group as an endogenous lipid carrier for the specific delivery of boron to different kind of tumours such as melanoma 20 , pulmonary breast metastases 21 and recently mesothelioma 22 . AT101 (Scheme 1) is a dual agent containing a carborane moiety functionalised with a Gd-based contrast agent. Among boron derivatives, carboranes occupy a special position both for their high boron content (10 B atoms) and their chemical versatility coupled with a high in vivo stability [23][24][25][26] .
In this study, PLGA nanoparticles functionalized with unique amino acid sequence consisting of 15 histidine (His) residues (CGGH 15 βA), namely PLGA-His, have been developed for the controlled release of the B-containing agent in the proximity of the tumour microenvironment with a pH triggered mechanism. In fact, the pKa of histidine in its oligomeric form is approximately 6.8 27 , thus being below pH 7 the imidazole ring is protonated. This occurs also when the oligo-His is conjugated to a PLGA polymeric chain forming a stable nanoparticle 28 . This was demonstrated thanks to the presence of 14 N atoms of histidine causing a quadrupolar relaxation enhancement (also called Quadrupolar Peak, QP) at 1.39 MHz, well detectable using a Fast Field Cycling NMR relaxometer 28 . The relaxation enhancement is pH dependent in the range 6.5-7.5, thus it acts as a reporter of the nanoparticle or scaffold integrity 28,29 . As it has been demonstrated that in the cancer microenvironment the pH is altered and more acidic than the physiological one 30 , herein, we propose the use of these B and Gd containing pH-sensitive nanoparticles which selectively interact with cell membranes to release the encapsulated drug in the proximity of tumour tissues. The use of poly-histidine conjugated polymers hybrid materials for pH Scheme 1. Schematic representation of PLGA-His nanoparticles. Oligo-His-PLGA was prepared according to the previously reported procedure 28 . Briefly, peptide sequence CGGH 15 βA was assembled by automated solid phase peptide synthesis and then conjugated to a PLGA-PEG 2 -Mal, prepared in-house from PLGA Resomer ® RG 502 H and Maleimide-PEG 2 -NH 2 . MRI. T 1 weighted images: glass capillaries containing cells pellets of untreated or treated with NPs (0.0974 mM [Gd]) AB22 and MeT-5A were placed in an agar phantom and acquired by MRI using a Bruker Avance Neo 300 MHz spectrometer (7 T) equipped with a Micro 2.5 microimaging probe (BrukerBioSpin, Ettlingen, Germany). T 1W images were obtained using a standard MSME (Multi-Slice Multi-Echo) sequence with the following parameters: TR = 250 ms, TE = 3 ms, FOV = 12 × 12 mm, slice thickness = 1 mm, NEX = 6, matrix size 128 × 128. Clonogenic assay. Clonogenic assay of irradiated and non-irradiated treated and untreated cells was performed seeding around 200 cells from each different flask in 6 cm diameter culture dishes. The growing of cells was followed for 18 days. The medium was renewed after 2-3 days. After 18 days, the cells were washed with PBS, fixed in methanol (25′) and stained with 0.5% (w/v) Crystal Violet in 20% ethanol (30′). The crystal violet was removed carefully and rinsed with tap water. Finally, the number of colonies was calculated by ImageJ software. The % was calculated by setting the irradiated control cells to 100%.

Results and discussion
PLGA nanoparticles preparation and characterisation.. The conjugation of PLGA with the oligo-His (Oligo-His-PLGA) was carried out following an already reported procedure 28 , based on a thiol-Michael addition reaction between PLGA-PEG 2 -maleimide and an oligo-His containing a N-terminal cysteine residue (amino acid sequence: CGGHnβA, n = 15). PLGA-NPs were prepared following the o/w emulsion solvent extraction method 38 using 100% of the commercially available PLGA (MW = 7-17 kDa; named PLGA-CTRL) or mixing 50% of this PLGA with 50% of Oligo-His-PLGA (PLGA-His). The theranostic agent AT101 (Scheme 1) was loaded into the NPs by adding it to the organic phase containing PLGA polymers. After dialysis the amount of AT101 remaining encapsulated with a very good yields (> 82%), was determined by measuring both B and Gd by ICP-MS. The average hydrodynamic diameters of PLGA nanoparticles, the millimolar relaxivity and the average surface-potential of the nanoparticles are reported in Table 1. The low and slightly negative Z potential observed in both NPs is due to the shielding effect of the negative charges of PLGA caused by both PVA and DSPE-PEG coating 39,40 . For the same reason only a slight Z potential increase was measured on PLGA-His at pH < 6.5. Moreover, part of the protonated oligo-His may remain in the particle core thus reducing their contribution to the measured Z potential ( Table 1). The nanoparticles were stored at 4 °C and their size was measured by DLS every 15 days. The results are reported in Table 2.
The relaxivity (r 1p ) is an expression of the contrast agent efficiency and corresponds to the paramagnetic contribution to the longitudinal relaxation rate normalized to the mM concentration of the Gd-complex solution. Both PLGA-CTRL and PLGA-His show a 4-5 times higher r 1p value (ca. 18 s −1 mM −1 , at 21.5 MHz and 25 °C, Table 1) with respect to those one observed with clinically used Gd-based contrast agents (in the range 4-5 mM −1 s −1 ) 41 . This is an indication of the long tumbling time filled by the AT101 complexes when loaded in the NP and the enhanced sensitivity reduces the detection limit of these imaging probes when internalized in a NP. The stability at 37 °C of the delivery system was assessed by monitoring for 72 h the relaxation rates of PLGA-NPs solutions at both physiological and tumoral pH, in a saline buffer (Hepes-NaCl buffer). To this purpose, solutions containing PLGA-CTRL and PLGA-His were stirred at 37 °C, both at pH 7.4 and 6.0, for 72 h under dialysis (cut-off 14 KDa). The relaxation rates of the PLGA solution in the dialysis membrane were  Fig. 1 shows that PLGA-CTRL are essentially stable for 72 h in these conditions, in the case of PLGA-His, there was observed an increased R 1 (s −1 ) when incubated at acidic pH, due to the protonation of the imidazole groups present in the NPs, with a consequent alteration of its physicochemical structure producing a swelling of the NP that facilitates water permeation. The swelling of the NP was confirmed by measuring the size of both PLGA-CTRL and PLGA-His after 24 h incubation at pH = 6.0 and 37 °C. In fact, despite the hydrodynamic diameter measured on PLGA-CTRL remained almost unchanged (149.0 ± 4 nm) the measured diameter of PLGA-His at pH 6.0 (160.4 ± 3.2 nm) increased of about 12% thus confirming the nanoparticle swelling and increase in volume. Moreover, as reported in the introduction oligo-histidine chains show a characteristic relaxation peak at 1.38 MHz due to the 14 N nuclear quadrupole resonance frequency of the imidazole groups present in the oligomeric chain, well distinct from the QPs generated by the background tissue. As the 14 N Quadrupolar Relaxation peak may be only observed for immobilized systems, changes in the intensity of the imidazole QPs can act as a reporter of the physical state of the poly-His solid/ liquid status and swelling related to the pH of the microenvironment. In reference 28 it was shown that the protonation of the imidazole group of the histidine (pKa = 6.8) increased the particle swelling and polymer solubility with a consequent increase in mobility and progressive disappearance of the QPs at pH < 6.0. Unfortunately, the QPs were not detectable in PLGA-His used in this work as the loaded paramagnetic Gd complex increases dramatically the relaxation rate becoming the predominant relaxation process.
In order to confirm this hypothesis, the Gd concentration was measured in the solutions inside the dialysis membrane (diluted 1:1 with buffer) after 0, 24 and 72 h incubation at 37 °C at both pH 7.4 and 6. The results reported in Table 3 shows that only at pH 6 a significant AT101 release was observed after 24 and 72 h incubation thus confirming the reduced stability of the nanoparticle at this pH.
Nanoparticle uptake in mesothelioma and healthy mesothelium cell lines. Uptake studies were performed using AB22 murine mesothelioma and, as a control, MeT-5A human healthy mesothelium cell lines, respectively. The two cell lines were incubated for 24 h with increasing Gd concentrations of both PLGA-His and PLGA-CTRL and after 24 h the amount of internalized Gd and B were measured by ICP-MS and normalized to the total cell proteins. Figure 2 shows that the nanoparticle uptake by AB22 tumour cells appears to be overall increased compared to the healthy mesothelial MeT-5A cell line. Moreover, the uptake of the oligo-His coated nanoparticles seems to be furtherly enhanced in mesothelioma cell line AB22 compared to the unconjugated nanoparticle. In conclusion, the presence of histidine in PLGA-His does not affect the uptake of the NPs in healthy cells suggesting a specific pH triggered mechanism occurring only in malignant cells, making these NPs suitable to specifically target this aggressive type of tumour cells.
From the data obtained by ICP-MS analysis, by converting mg of proteins in number of cells (1 mg of protein correspond to 1.7 and 6.3 million cells for AB22 and MeT-5A, respectively) and assuming that in the case of epithelial tumours (with a diameter ranging 15-20 μm), a density of ca. 10 8 cells for cm 3 is a reasonable number for these tumour tissues 42 , it was possible to calculate the maximum ppm of B internalised in cells, reported in Table 4.  www.nature.com/scientificreports/ Subsequently, in order to assess if the concentration of the internalized AT101 was sufficient for the generation of a detectable MRI Signal Intensity enhancement, a T 1 weighted image was acquired on AB22 and MeT-5A cells incubated with PLGA-CTRL or PLGA-His, both at 0.097 mM (Gd concentration) and after washing, centrifuging the cells at the bottom of glass capillaries. As we can observe in Fig. 3, the uptake of the nanoparticles is consistent with the previous findings, suggesting an increased uptake of the PLGA-His in mesothelioma cells compared to healthy ones and also compared to the cells incubated with PLGA-CTRL.
Neutron cell irradiation. Cells incubated with PLGA nanoparticles encapsulating AT101 were irradiated with thermal neutrons at the Triga Mark II nuclear reactor at the University of Pavia. For this set of experiments, AB22 and MeT-5A cells were pre-incubated for 24 h in three different conditions: (1) with medium only (CTRL cells), (2) with PLGA-CTRL and (3) with PLGA-His. PLGA-NPs were incubated at a concentration corresponding to a 0.097 mM Gd concentration in the cell medium. Cells were irradiated at the thermal column for 15′ with a reactor power of 30 kW. As shown in Fig. 4A, a strong cytotoxic effect of BNCT is clearly detected on AB22 cells, with a significant decrease in the number of cells 24 h after irradiation. In fact, after irradiation, compared to the irradiated control group, 30% of the AB22 cells treated with PLGA-CTRL died, and regarding PLGA-His, the percentage increases to 60%. On the contrary, MeT-5A cells showed no significant decrease 24 h after irradiation, consistent with the lower NPs uptake of this cell type (as shown in Fig. 2 and Table 2). This observation   www.nature.com/scientificreports/ is supported by the results obtained with clonogenic assay carried out at day 18 after irradiation. In fact, Fig. 4B shows that AB22 after irradiation, in particular if treated with PLGA-His, stop to form colonies. These results suggest a significant decrease in the ability of mesothelioma cells to reattach and when treated with PLGA NPs, particularly effective in the case of PLGA-His administration. On the contrary, MeT-5A healthy cells show only a slightly reduced colony formation with respect to irradiated untreated control cells.

Conclusions
Mesothelioma is an aggressive cancer that is associated with asbestos exposure. Although asbestos is banned in several countries, an epidemic of mesothelioma is predicted to affect middle-income countries during this century owing to their heavy consumption of asbestos. Patients affected by this disease often show a poor prognosis because of the lack of significant and effective therapies since conventional chemotherapy fails to affect and eradicate this cancer subtype effectively 43 . Although recent work has found significant possibilities for treatment, identifying genetic and pathophysiological vulnerabilities, mesothelioma remains one of the cancers with the lowest survival rate. Therefore, in this paper, we ought to demonstrate the possibility of an innovative approach, based on Boron Neutron Capture Therapy, with a theranostic compound delivered by PLGA nanoparticles. We developed and characterised PLGA nanoparticles containing poly-histidine chains, able to retain the theranostic compound AT101 effectively and deliver it specifically to the tumour cells, thus taking full advantage of this pre-targeted radiotherapy. It was demonstrated in this paper how these nanoparticles are preferentially taken-up by mesothelioma than healthy mesothelial cells, suggesting the possibility for a targeted therapeutic approach in the cure of this dreadful disease. Consequently, AB22 mesothelioma cells were strongly affected by neutron irradiation causing their irreversible damage, whereas a high percent of healthy cells was spared by the same